Anti-Inflammatory and Phytochemical Analysis of the Crude Leaves Extracts of Boscia Coriacea Graells and Uvaria Leptocladon Oliv

Background The objective of this study was to evaluate the anti-inflammatory activities and phytochemical composition of the leaves extracts of Boscia coriacea Graells and Uvaria leptocladon Oliv. Methods The powdered leaves of Boscia coriacea Graells and Uvaria leptocladon Oliv were extracted by maceration and soxhlet extraction methods. Anti-inflammatory activity of the leaves extracts of Boscia coriacea Graells and Uvaria leptocladon Oliv were evaluated using carrageenan-induced paw edema model. Standard methods were used for analysis of phytochemical composition of the leaves extracts of Boscia coriacea Graells and Uvaria leptocladon Oliv. Data analysis was done using one way analysis of variance. Results U. leptocladon Oliv (200 mg/kg) and B. coriacea Graells (200 mg/kg) showed percent inhibition on mice paw edema of 86% and 75% after six hours of carrageenan injection, respectively. The ethanol fraction (100 mg/kg) of U. leptocladon Oliv showed the highest anti-inflammatory effect after six hours of carrageenan injection. The phytochemical analysis of the leaves extracts of B. coriacea and U. leptocladon revealed the presence of tannins, alkaloids, cardiac glycoside, flavonoids, phenols, quinones, and saponins. Conclusion The crude leaves extracts of B. coriacea Graells and U. leptocladon Oliv contain phytochemicals with anti-inflammatory activities.


INTRODUCTION
Inflammation is one of the mechanisms of immune response that destroys infectious agents. However, uncontrolled inflammatory response may cause chronic diseases, including diabetes mellitus, cancer, arteritis, cardiovascular and vascular diseases (1). Prolonged use of steroidal anti-inflammatory drugs (SAIDs) and non-steroidal anti-inflammatory drugs (NSAIDs) that are used for the treatment of inflammatory conditions have been shown to cause numerous Therefore, it is necessary to search for safe and effective anti-inflammatory drugs.
Medicinal plants are major sources of chemicals with anti-inflammatory activities. Some medicinal plants that belong to the genus Uvaria and Boscia have anti-inflammatory properties (4,5). The genus Boscia belongs to the family Capparidaceae (6), which is widely distributed in Ethiopia. It is found in Bale, Sidamo, Kefa, Konso, Gamo Gofa and Hararge regions of Ethiopia (7). The genus Uvaria belongs to the family Annonaceae that is also widely distributed in Ethiopia. For example, Uvaria leptocladon is found in Sidamo, Kefa, Konso (Alie), and Gamo Gofa regions of Ethiopia (8). Thus, the objective of this study was to evaluate the anti-inflammatory activities and phytochemical composition of the crude leaves extracts of B. coriacea Graells and U. leptocladon Oliv.

MATERIALS AND METHODS
Plant collection: Fresh leaves of B. coriacea Graells and U. leptocladon Oliv were collected in April 2021 from Alie and Konso, which are located in Southern Ethiopia. After the plant materials were authenticated by one of the authors, a Botanist at the Department of Plant Biology and Biodiversity Management, a voucher specimen of each plant (ST001 and ST002, representing B. coriacea Graells and U. leptocladon Oliv, respectively) was deposited at the National Herbarium, College of Natural and Computational Sciences, Addis Ababa University. Then, the leaves samples were cleaned and allowed to dry in shade avoiding direct sun light.

Extraction:
The powdered leaves of B. coriacea Graells (100 g) and U. leptocladon Oliv (100 g) were extracted by soxhlet extraction method using a liter (1 L) of 80% methanol (MeOH). In addition to this, the powdered leaves of B. coriacea Graells (1 kg) and U. leptocladon Oliv (1 kg) were extracted by maceration method using 10 liters of 80% MeOH. Then, the mixture was filtered using Whatmann no.1 filter paper. The MeOH was removed from the filtrate by evaporation using rotary evaporator. The water component of the mixture was removed by lyophilization using a lyophilizer equipment (Christ, ALPHA 2-4-LD Plus). The 80% MeOH extract was fractionated using chloroform (CHCl3), MeOH and CHCl3-MeOH (1:1) in the Department of Chemistry, College of Natural and Computational Sciences, Addis Ababa University. The extraction yields (w/w) were 5% and 18% for maceration and soxhlet extracts, respectively. All the extracts were stored at -20°C until experiments were conducted.

Evaluation of anti-inflammatory activity:
The anti-inflammatory activities of the leaves extracts of B. coriacea Graells and U. leptocladon Oliv were evaluated by carrageenan induced paw edema model (9). First, the Swiss Albino mice were randomly grouped into three groups, each group containing six mice. Thirty minutes before carrageenan injection, the first group was given leaf extract and the second and third groups were orally treated with positive control (indomethacin) and negative control (normal saline). After thirty minutes, 50 µl of 1% freshly prepared solution of carrageenan was injected into the left-hind paw of each mouse. For each mouse, the initial diameter of the left hind paw was measured using micrometer (Digimatic micrometer, Mitu Toyo Corporation) before administration of the extracts. The diameter of paw was also measured post-carrageenan injection at time zero (just after carrageenan injection), and every hour for six consecutive hours (1h, 2h, 3h, 4h, 5h and 6h after carrageenan injection).
Percentage reduction of paw edema was calculated using the following formula:-(Vt-V0 in control group)-(Vt-V0 in treatment group) x100 Vt-V0 in control group Where, V0 = is the mean diameter of paw before injection of carrageenan in treated and negative control groups and Vt= is the mean diameter of paw in treated and negative control groups after carrageenan injection at time t.
Ethical considerations: Ethical clearance was obtained from the Institutional Review Board of the College of Natural and Computational Sciences of Addis Ababa University. Copy is attached as additional file. Data analysis: Analysis of differences between anti-inflammatory activities of the fractions of U. leptocladon Oliv leaf extract was done by using one way analysis of variance with post hoc comparison (Tukey's test).

Anti-inflammatory activity of the leaves extracts of B. coriacea Graells and U.
leptocladon Oliv: The leaves extracts of both B. coriacea Graells and U. leptocladon Oliv showed anti-inflammatory effects in carrageenan-induced paw edema model. U. leptocladon Oliv (200 mg/kg) and B. coriacea Graells (200 mg/kg) showed percentage inhibition on mice paw edema of 86% and 75% after six hours of carrageenan injection, respectively ( Table 1). As shown in Table 2, the ethanol fraction (100 mg/kg) of the leaf extract of U. leptocladon Oliv demonstrated the highest (77.7%) anti-inflammatory effect after six hours of carrageenan injection as compared with ethyl acetate (50%) and chloroform (66.7%) fractions.

DISCUSSION
The anti-inflammatory activity of medicinal plants is related to the presence of phytochemicals such as saponins, alkaloids, tannins, cardiac glycosides, and flavonoids (14,15). The anti-inflammatory activities of the crude leaves extracts of B. coriacea Graells and U. leptocladon Oliv observed in this study might be due to the presence of saponins, alkaloids, cardiac glycosides, flavonoids, and tannins in the leaves extracts. The anti-inflammatory activity of the U. leptocladon Oliv observed in this study is comparable with the findings of other studies that used carrageenan-induced paw edema model to assess the anti-inflammatory activity of the leaf extract of Uvaria chamea (4,16). The highest antiinflammatory activity of the ethanol fraction of U. leptocladon Oliv leaf extract suggests that the inflammatory constituents are contained in this fraction. Acute inflammation induced by carrageenan is linked with the induction of production of inflammatory mediators such as histamines, serotonins, bradykinin and prostaglandins (17). Therefore, the inhibition of paw edema in the mice that were administered with the leaves extracts of B. coriacea Graells and U. leptocladon Oliv could be due to the inhibition of production of serotonin, histamine, bradykinin and prostaglandins by the phytochemicals that are found in the leaves extracts of B. coriacea Graells and U. leptocladon Oliv.
The phytochemical constituents of the leaves extracts of U. leptocladon Oliv and B. coriacea Graells identified in this study is also comparable with the findings of other researchers that investigated the phytochemical composition of the leaves extracts of other plants that belongs to the genus Uvaria such as U. chamae (4,16,18,19) and the genus Boscia such as B. senegalensis, B. arabica and B. variabilis (20,21).